CRD and technical validation of ImmunoCAP ISAC ImmunoCAP ISAC Customer days Phn. Biol. E. Cavalier Phn. R. Gadisseur 19/11/2009

CRD and technical validation of ImmunoCAP ISAC

ImmunoCAP ISAC Customer days

Phn. Biol. E. Cavalier

Phn. R. Gadisseur

19/11/2009

Timeline…allergy diagnosis

1880 1995-991988-911967 2000 2008

Provocation tests (SPT)

First allergens

clonedRAST

Characterization & designation of

IgE

Recombinant allergen panels recreating allergen extracts

First allergen microarray

ImmunoCAP ISAC (Phadia/ VBC Genomics)

IN-VITRO testing

IN-VIVO testing

Component – Resolved -Diagnostics

Standardization of allergen extracts

Components : Production

Components - Resolved Diagnosis (CRD)

« CRD »

Component-Resolved Diagnosis uses defined recombinant allergens to dissect the individual patient’s IgE reactivity profile with the aim of identifying the

disease-eliciting molecules. Valenta R et al, Clin Exp Allergy 1999

Molecules, genuine markers tell you the truth !

Mari A., VIP Customers day, Brussels, 17th November 2009.

The presence of IgE to cross-reactive allergen components can be determined and used to predict clinically relevant sensitization to allergen sources which contain immunologically

related allergens.

Allergens, data bases

Peanut allergens Biochemical property of the allergens

« specific » IgE for extracts

• From a laboratory point of view… – What does one value of sIgE means for allergen extracts ?

– How to manage this sIgE rate ?

– Concerning the care of the patient ?

– Concerning the risks ? Impact on overall patient management and health outcomes (severity

assessment, risk, therapy choices, improve patient’s quality of live).

• Is there an interest of Component-resolved diagnosis in the laboratory routine practice?– How can we realize CRD in the laboratory?

Our experience with peanut sensitization…– + sIgE for f13, anamnesis, components

– Our question is :”Should we recommend the strict avoidance of peanut ?”

Clinical Cases (I)

1. Julie, F : f13 (peanut) = 27.4 kUA/L. SPT + : peanut, walnut, hazelnut, sesame seed, weeds,

birch, pine nut. SPT - : soybean, egg white et walnut. Asthma, gastrointestinal intolerance and erythema after

ingestion of nuts.

Clinical cases (II)

2. Laurent, H : f13 (peanut) = 1.22 kUA/L. sIgE + : birch, apple, pear, peach, apricot, hazelnut, walnut and recently to

celery and to fennel. Atopy, polysensitised, oral allergy syndrome (OAS) and a recent evolution to

breathing difficulties.

3. Maud, F : f13 (peanut) = 1.23 kUA/L sIgE + : pollens of weeds, cow milk, carrot, orange, tomato, wheat, potato,

garlic, onion, celery, banana, peach and peanut. Sensitization to a wide variety of foods from plants. Gastro-intestinal disorders.

Clinical cases (III)

4. Eddy, M :

f13 (peanut) = 0,44 kUA/L SPT+ : birch, apple, peach, banana, garlic, peanut Swollen lips

Sensitization to birch…What can we conclude?

Clinical cases (IV)

5. Nadia, F : f13 (peanut) = 1,73 kUA/L f17 (hazelnut) = 4,23 kUA/L

sIgE + : apple, peach, hazelnut, peanut SPT +++ : peanut, hazelnut, walnut. Atopy, polyallergy, rhinoconjonctivitis, OAS, urticaria et

angiœdema

In those 5 clinical cases…

sIgE rates for peanut extracts are they proportional to the gravity of the allergy ?

What are the risks for those patients?

Eviction of diet or not ?

How to manage the patient?

What will be the future of the patient ?

?

Predict cross reactions

• Storage protein– Protein found in seeds serving as source material during the

growth of a new plant

– Often stable and heat-resistant proteins causing reactions also to cooked foods

rBer e 1

Ara h 1 Ara h 2 Ara h 3

Cor a 9

rSes i 3

nGly mβ-conglycininnGly m glycinin


• PR-10 protein, Bet v 1 homologue– A heat labile protein, cooked foods are often tolerated.

» Breiteneder H, Biotechnol Adv, 2005

– Often associated with local symptoms such as oral allergy syndrome (OAS).

– Often associated with allergic reactions to fruits, vegetables and spices in northern Europe.

Mal d 1

Dau c 1

Pru p 1

Ara h 8

Cor a 1.01 Cor a 1.04

Api g 1Gly m 4

Bet v 1

Pru ar 1Pyr c 1 Pru av 1

Oral Allergy Syndrome (OAS)

• Oral Allergy Syndrome : allergic reactions that occur rapidly, within minutes of eating a trigger food.

– An itching or burning sensation in the lips, mouth, and/or pharynx.

• Associated to a sensitization in one or more proteins like PR-10 (Bet v 1-like) (birch).

1 60 Birch MGVFNYETET TSVIPAARLF KAFILDGDNL FPKVAPQAIS SVENIEGNGG PGTIKKISFP Hazel MGVFNYETES TSVIPAARLF KAFILDGNNL IPKVAPQAVS SVENVEGNGG PGTIKKITFS Alder MGVFNYEAET PSVIPAARLF KAFILDGDKL LPKVAPEAVS SVENIEGNGG PGTIKKITFP Hornbeam MGVFNYEAET TSVIPAARLF KAFILDGNNL IPKVAPQAVS SVENVEGNGG PGTIKKITFS Beech MGVFTYESEN TSVIPPARLF KAFVLDADNL IPKVAPQSIK STETLEGDGG PGTIKKITFG Apple MGVFNYETET TSVIPAPRLF KAFILDGDNL IPKIAPQAIK STEIIEGDGG VGTIKKVTFG Cherry MGVFTYESEF TSEIPPPRLF KAFVLDADNL VPKIAPQAIK HSEILEGDGG PGTIKKITFG Apricot MGVFTYETEF TSVIPPEKLF KAFILDADNL IPKVAPTAVK GTEILEGDGG VGTIKKVTFG Pear MGLYTFENEF TSEIPPPRLF KAFVLDADNL IPKIAPQAIK HAEILEGNGG PGTIKKITFG Medicago MGVFNFEDET TSIVAPARLY KALVTDSDNL IPKVI.DAIQ SIEIVEGNGG AGTIKKLTFVGarden pea MGVFNVEDEI TSVVAPAILY KALVTDADNL TPKVI.DAIK SIEIVEGNGG AGTIKKLTFV Soybean MGVFTFEDEI NSPVAPATLY KALVTDADNV IPKAL.DSFK SVENVEGNGG PGTIKKITFL Tomato MGVNTYTYES TTTISPTRLF KALVLDFDNL VPKLLSQHVK NNETIEGDGG VGSIKQMNFV Celery MGVQKTVVEA PSTVSAEKMY QGFLLDMDTV FPKVLPQLIK SVEILEGDGG VGTVKLVHLG Carrot MGAQSHSLEI TSSVSAEKIF SGIVLDVDTV IPKAAPGAYK SVDV.KGDGG AGTVRIITLP Asparagus MSSSAVSHEI ESSVSAARLF KASMIEWHNL APKILPEIVS SASVVAVDGG VGSIRQINFT

120 Birch EGFPFKYVKD RVDEVDHTNF KYNYSVIEGG PIGDTLEKIS NEIKIVATPD GGSILKISNK Hazel EGSPFKYVKE RVEEVDHTNF KYSYTVIEGG PVGDKVEKIC NEIKIVAAPD GGSILKISNK Alder EGSPFKYVKE RVDEVDRVNF KYSFSVIEGG AVGDALEKVC NEIKIVAAPD GGSILKISNK Hornbeam EGSPVKYVKE RVEEVDHTNF KYSYTVIEGG PVGDKVEKIC NEIKIVAAPD GGSILKITSK Beech EGSQFKYVKH RIDEVDQANF SYGYSVIEGD VVSGIIEKIS YEIKIVASPD GGSLLKSTSK Apple EGSQYGYVKQ RVNGIDKDNF TYSYSMIEGD TLSDKLEKIT YETKLIASPD GGSIIKTTSH Cherry EGSQYGYVKH KIDSIDKENY SYSYTLIEGD ALGDTLEKIS YETKLVASPS GGSIIKSTSH Apricot EGSQYAYVKH RVDGIDKDNL SYSYTLIEGD ALSDVIENIA YDIKLVASPD GGSIVKTTSH Pear EGSQYGYVKH RVDSIDEASY SYAYTLIEGD ALTDTIEKIS YEAKLVASGS .GSTIKSISH Medicago EGGETKYDLH KVDLVDDVNF AYNYSIVGGG GLPDTVEKIS FESKLSAGPD GGSIAKLTVKGarden pea EDGETKHVLH KVELVDVANL AYNYSIVGGV GFPDTVEKIS FEAKLSAGPN GGSIAKLSVK Soybean EDGETKFVLH KIESIDEANL GYSYSVVGGA ALPDTAEKIT FDSKLVAGPN GGSAGKLTVK Tomato EGGPIKYLKH KIHVIDDKNL ETKYSLIEGD ILGEKLESIT YDIKFEANDN GGCVYKTTTE Celery EATEYTTMKQ KVDVIDKAGL AYTYTTIGGD ILVDVLESVV NEFVVVPT.D GGCIVKNTTI Carrot EGSPITSMTV RTDAVNKEAL TYDSTVIDGD ILLGFIESIE THLVVVPTAD GGSITKTTAI Asparagus SAMPFPYLKE RLDFVDEANF ECKSSLIEGG DLGTKLESAS SHFKLVPSSN GGCVVKLEGI

178 Birch YHTKGDHEVK AEQVKASKEM GETLLRAVES YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Hazel YHTKGDHEVD AEHIKGGKEK VEGLFRAVEA YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Alder FHTKGDHEIN AEQIKIEKEK AVGLLKAVES YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Hornbeam YHTKGDHEVP AEHIKGGKER VEGLLKPVEA YLLAHTAEYN N~~~~~~~~~ ~~~~~~~~ Beech YHIKGNHEIK EEEVKAGKEK AAGLFKAVEA YLLAHPDAYN ~~~~~~~~~~ ~~~~~~~~ Apple YHAKGDVEIK EEHVKAGKEK ASGLFKLLEA YLLAHSDAYN ~~~~~~~~~~ ~~~~~~~~ Cherry YHTKGNVEIK EEHVKAGKEK ASNLFKLIET YLKGHPDAYN ~~~~~~~~~~ ~~~~~~~~ Apricot YHTKGDVEIK EEQVKAGKEK AAGLFKLVEA YLLANPDAYN ~~~~~~~~~~ ~~~~~~~~ Pear YHTKGDIEIK EEHVKAGKEK AHGLFKLIES YLKDHPDAYN ~~~~~~~~~~ ~~~~~~~~ Medicago YFTKGDAAPS EEEIKGGKAR GDGLFKALEG YVLANPD.Y~ ~~~~~~~~~~ ~~~~~~~~Garden pea YFTKGDAAPS EEQLKTDKAK GDGLFKALEG YCLAHPD.YN ~~~~~~~~~~ ~~~~~~~~ Soybean YETKGDAEPN QDELKTGKAK ADALFKAIEA YLLAHPD.YN ~~~~~~~~~~ ~~~~~~~~ Tomato YHTKGDHVVS EEEHNVGRRE NHEYFQGCRS IPSRESFCLR LNIDEKESGL HVRNYACT Celery YNTKGDAVLP EDKIKEATEK SALAFKAVEA YLLANLQFLA ~~~~~~~~~~ ~~~~~~~~ Carrot FHTKGDAVVP EENIKFADEQ NTALFKAIEA YLIAN~~~~~ ~~~~~~~~~~ ~~~~~~~~ Asparagus FKALPGVETT DEVAK.GKEM MTNAIKAAEA YLVANPTAYA ~~~~~~~~~~ ~~~~~~~~

o Hazelnut, 46%o Apple, 39%o Peach, 24%o Cherry, 22%o Nut, 21%o Pear, 20%o Almond, 19%o Plume, 17%

o Brazil nut, 16%

o Potato, 15%

o Carrot, 13%

o Peanut, 10%

o Strawberry, 10%

o Orange, 9%

o Apricot, 7%

PR-10 rBet v 1rPru p 1rGly m 4rAra h 8

rApi g 1.01rAln g 1rCor a 1nAct d 8rMal d 1rDau c 1


• LTP (non-specific Lipid Transfer Protein, nsLTP)– A protein stable to heat and digestion causing reactions also to

cooked foods

– Often associated with systemic and more severe reactions in addition to OAS

– LTPs often cause food allergy to fruit in the absence of pollen allergy.

– Often associated with allergic reactions to fruit and vegetables in southern Europe.

Cor a 8 Pru p 3

Ara h 9

Mal d 3

Zea m 14

Pru av 3

LTP nPru p 3rCor a 8 rPar j 2nArt v 3Ara h 9Mal d 3Zea m 4Jug r 3

Pru ar 3


• Profilin– Seldom associated with clinical symptoms but may cause

demonstrable or even severe reactions in a small minority of patients

– Profilins are present in a broad range of pollens and foods including trees, grasses and weeds.

– There is a wide range of homology between profilins from different fruits and vegetables.

rMal d 4

rHev b 8

rPhl p 12rBet v 2

rMer a 1

Ara h 5

Pru p 4

rCor a 2 rOle e 2 rPyr c 4

PFL rPhl p 12

rBet v 2 nOle e 2 rMer a 1 rHev b 8

rMal d 4 rPru p 4

nAct d 9

nAna c 1 nAra h 5

To resume…

Local (OAS)

Characteristic symptoms

Systemic

Sensitization profiles

PR-10 proteins

ns-LTP Profilins

In-vitro cross-reactions

• CCD (MUXF3) :– Marker of sensitization to Carbohydrate determinants (cross-reacting

carbohydrate determinants). Glyco-epitopes. – Present in most plants,– sIgE directed towards glycans appear to show the widest pattern of

cross-reactivity among allergenic extracts and are often responsible for observed in-vitro cross-reactions.

« in-vivo » effects ? – Seldom associated to clinical symptoms …

» But, publications ? » To compare to the clinical informations and to SPT…

– Test if… Sensitization to fruits and vegetables, seeds. Sensitization to latex in a pollinic patient

without occupational risk exposure. Sensitization to Hymenoptera venom

with/without sensitization to pollens.

Arachis Hypogea

•Ara h 1 : Storage protein (vicillin)

• Major allergen

•Ara h 2 : Storage protein (conglutin)

• + frequent, sensitizing potential ++, stability ++ to digestion.

•Ara h 3 : Storage protein (glycinin)

• Minor allergen, rare sensitizations.

•Ara h 8 : PR-10

• Major allergen of peanut in birch pollen allergic patients.

• Sensitive to heat and digestion.

•Ara h 9 : nsLTP

• Stable to heat and digestion.

• Severe reactions >> systemic reactions !

•Ara h 8 : Profilin

• Cross-reactions between many fruits and vegetables.

•CCD (MUXF3)

• Cross-reactive Carbohydrate Determinants

• Seldom associated to symptoms.

• Present in most plants.

Allergenicity :

Ara h 2 is 100 times stronger than Ara h 1

Koppelman SJ et al, Clin Exp Allergy 2004,34:583

Ara h 2 possesses high proteolytical stability

Lehmann K et al, Biochem J, 2006:395-463

After roasting peanuts, allergenicity of Ara h 2 is multiplicated by 3

Maleki SJ et al, J Allergy Clin Immunol, 2003

Laboratory practice : How to diagnose peanut allergy ?

Peanut (f13) + Ara h 2 (f423)

Peanut (f13) : Neg

Ara h 2 : Neg

Low risks of severe reactions

How to explore ?

Recommendations

Birch (north Europe) : test Ara h 8 (f352)

Peanut (f13) : Pos

Ara h 2 : Neg

Risk of severe reaction?

How to explore? Recommendations

Test : Risk

• Ara h 1 (f422)

•Ara h 3 (f424)

•Ara h 9 (f427)

•Ara h 8 (f352)

•CCD (Ro214)

Peanut (f13) : Pos

Ara h 2 : Pos

Very high risk of severe reactions

Clinical Cases (1)

1. Julie, F : f13 (peanut) = 27.4 kUA/L.

– SPT + : peanut, walnut, hazelnut, sesame seed, birch, weeds, pine nut.

– SPT - : soybean, egg white et walnut.

Asthma, gastrointestinal intolerance and erythema after ingestion of nuts.

What are the risks ? Birch?

Peanut (f13) = 27,4 kUA/L

Ara h 2 (f423) = 14,2 kUA/L

Tests : Risks

• Ara h 1 = 3,46 kUA/L

•Ara h 3 = 0,6 UA/L

•Ara h 9 <0,1 kUA/L

•Ara h 8 = 2,27 kUA/L

•CCD < 0,1 kUA/L

Peanut (f13) : Pos

Ara h 2 : Pos

Major allergens of peanut

Storage proteins

peanut PR-10 protein !

• Sensitization to Ara h 8 due to birch allergy (PR-10).

• Sensitization to Ara h 2 : Storage protein (conglutin), Stable ++ to digestion, Diet eviction of peanut because risks of severe reactions +++.

• Sensitization to 3 major allergens of peanut.

Clinical cases (1)

Cross-reaction : storage proteins ??



Clinical cases (2)

2. Laurent, H : f13 (peanut) = 1.22 kUA/L.

– sIgE + : birch, apple, pear, peach, apricot, hazelnut, walnut and recently to celery and to fennel.

Atopy, polysensitised, oral allergy syndrome(OAS) and a recent evolution to breathing difficulties.

What are the risks?– What about the Birch?

– Recent evolution of the allergy?


Ara h 2 (f423) < 0,10 kUA/L

Peanut (f13) : Pos

Ara h 2 : Neg

Risk of severe reactions?

Tests : Risk

• Ara h 1 < 0,1 kUA/L

•Ara h 3 < 0,1 kUA/L


•Ara h 8 = 8,4 kUA/L

•CCD < 0,1 kUA/L

Clinical cases (2)

PR-10, Bet v 1-homologue

• Sensitization to peanut after birch allergy (PR-10 proteins).

• Sensitization to other PR-10 : - Apple (Mal d 1), pear (Pyr c1), peach (Pru p 1), apricot (Pru ar 1), hazelnut (Cor a 1) and recently to celery (Api g 1)

•In this case, evolution of the OAS with respiratory distress explained by recent sensitization to Api g 1.


Clinical cases (3)

3. Maud, F : f13 (peanut) = 1.23 kUA/L

– sIgE + : pollens of weeds, cow milk, carrot, orange, tomato, wheat, potato, garlic, onion, celery, banana, peach and peanut.

Sensitisation to a wide variety of foods from plants.

Gastro-intestinal disorders.

What are the risks ?– Multiple sensitizations to fruits and vegetables.


Ara h 2 (f423) < 0,1 kUA/L

Peanut (f13) : Pos

Ara h 2 : Nég

Tests : Risks

• Ara h 1 < 0,1 kUA/L




•CCD = 1,93 kUA/LCCD

Clinical cases (3)

• Sensitization to CCD : - Marker of sensitization to Carbohydrate Determinants. - Multiple cross-reactions. - Seldom associated to clinical symptoms.

• Test CCD if sensitization to fruits and vegetables, seeds.



Clinical cases (4)

4. Eddy, M : f13 (peanut) = 0,44 kUA/L

– SPT+ : birch, apple, peach, banana, garlic, peanut

Swollen lips

What are the risks ?– Sensitization to birch…


Ara h 2 (f423) < 0,1 kUA/L

Testés

• Ara h 1 < 0,1 kUA/L




•CCD < 0,1 kUA/L

?Bet v 2 = 4,6 kUA/L

Clinical Cases (4)

• Sensitization to profilins (Bet v 2)

• Seldom associated with clinical symptoms except OAS and rarely severe reactions in a minority of patients.

• Presence of profilins in many fruits and vegetables. - Birch (Bet v 2), apple (Mal d 4), peach (Pru p 4), banana (Mus a 1), garlic (All c 4), peanut (Ara h 5).

Peanut (f13) : Pos

Ara h 2 : Neg



Clinical cases (5)

5. Nadia, F : f13 (peanut) = 1,73 kUA/L f17 (Hazelnut) = 4,23 kUA/L

– sIgE + : apple, peach, hazelnut, peanut– SPT +++ : peanut, hazelnut, walnut.

Atopy, polyallergy, rhinoconjonctivitis, OAS, urticaria et angiœdema

What are the risks ? …angioedema..;

Clinical cases (5)


Ara h 2 (f423) <0,1 kUA/L

Peanut (f13) : Pos

Ara h 2 : Neg

Test : Risks

• Ara h <0,1kUA/L

•Ara h 3 <0,1kUA/L

•Ara h 9 = 8,2 kUA/L

•Ara h 8 <0,1kUA/L

•CCD <0,1kUA/L

nsLTP



Hazelnut (f17) = 4,23 kUA/L

Cor a 8 (f425) = 3,87 kUA/L

Hazelnut (f17) : Pos

Cor a 8 : Pos


nsLTP

•Sensitization to nsLTP : - Marker of cross-reactions.- Associated to severe reactions. - Occurs mainly in South of Europe.- Sensitization to apple (Mal d 3), peach (Pru p 3), hazelnut (Cor a 8) and peanut (Ara h 9).

More complex…

• Julien, M : – Complex clinical case !!

Atopy, Atopic dermatitis, Perannual rhinitis, Giant eczema. Food allergy (egg white, peanut,

soybean, crab and garlic)– How to evaluate the symptoms ?

SPT Pneumallergens:– + : D1 et D2, dog, weeds.– Suspiscious : birch.

SPT Trophallergens :– + White egg, peanut, soybean, garlic.

Peanut,f13 9.71

Soybean,f14 4.43

Crab,f23 1.98

Garlic,f47 4.11

Egg yellow,f75 0.61

Pityrosporum orbiculare,m70 3.93

Dermatophagoïdes pteronyssinus,d1 >10

Egg white,f1 5.37

Schrimp ,f24 2.64

Gx3:g1+g5+g6+g12+g13 93.6

Birch,t3 58.0

IgEs for extracts

Thanks to all the recombinants,

We would be able to explain everything !!

Request form ImmunoCAP™Components

Grass pollens : g6 Phleum pratense rPhl p 1 Phleum pratense g205 rPhl p 2 Phleum pratense g206 nPhl p 4 Phleum pratense g208 rPhl p 5b Phleum pratense g215 rPhl p 6 Phleum pratense g209 rPhl p 7 Calc. Bind. Prot. Phleum pratense g210 rPhl p 11 Phleum pratense g211 rPhl p 12 Profilin Phleum pratense g212 Mix rPhl p 1, rPhl p 5b Phleum pratense g213 Mix rPhl p 7, rPhl p 12 Phleum pratense g214 Weed pollens : w6: Artemisia vulgaris nArt v1 Artemisia vulgaris w231 Tree pollens : t3: Betula verrucosa rBet v 1 PR10 Betula verrucosa t215 rBet v 2 Profilin Betula verrucosa t216 rBet v 4 Calc. Bind. Prot. Betula verrucosa t220 rBet v 6 Betula verrucosa t225 Mix rBet v 2, rBet v 4 Betula verrucosa t221 t9: Olea europaea nOle e 1 Olea europaea t224 Mites d1: Dermatophagides pteronyssinus nDer p1 Dermatophagides pteronyssinus d202 rDer p2 Dermatophagides pteronyssinus d203 rDer p10 Tropomyosin Dermatophagides pteronyssinus d205 Microorganisms m3 : Aspergillus fumigatus rAsp f 1 Aspergillus fumigatus m218 rAsp f 2 Aspergillus fumigatus m219 rAsp f 3 Aspergillus fumigatus m220 rAsp f 4 Aspergillus fumigatus m221 rAsp f 6 Aspergillus fumigatus m222 m6 : Alternaria alternata rAlt a 1 Alternaria alternata m229

Epidermals & other proteins e4: Cow Bos spp nBos d 6 BSA Serum albumin Bos spp. e204 e5: Dog Canis familiaris rCan f 1 Canis familiaris e101 rCan f 2 Canis familiaris e102 nCan f 3 Serum albumin Canis familiaris e221 e1: Cat Felis domesticus rFel d 1 Felis domesticus e94 nFel d 2 serum albumin Felis domesticus e220 e83: Swine Sus scrofa Swine serum albumin Sus scrofa e222 Foods f85: Celery Apium graveolens rApi g 1.01 PR10 Apium graveolens f417 f13: Peanut Arachis hypogaea rAra h 1 Arachis hypogaea f422 rAra h 2 Arachis hypogaea f423 rAra h 3 Arachis hypogaea f424 rAra h 8 PR10 Arachis hypogaea f352 rAra h 9 LTP Arachis hypogaea f427 f17: Hazelnut Corylus avellana rCor a1 PR10 Corylus avellana f428 rCor a8 LTP Corylus avellana f425 f18: Brazil nut Bertholletia excelsa rBer e 1 Bertholletia excelsa f354 f14: Soy Glycine max rGly m 4 PR10 Glycine max f353 f95: Peach Prunus persica rPru p 1 PR10 Prunus persica f419 rPru p 3 LTP Prunus persica f420 rPru p 4 Profilin Prunus persica f421 f4: Wheat Triticum spp rTri a 19; Omega-5 Gliadin Triticum spp. f416

f1 Egg white nGal d 1 Ovomucoid Gallus spp. f233 nGal d 2 Ovalbumin Gallus spp. f232 nGal d 3 Conalbumin Gallus spp. f323 nGal d 4 Lysozyme Gallus spp. k208 f2: Milk Bos spp nBos d 4 α-lactalbumin Bos spp. f76 nBos d 5 β -lactoglobulin Bos spp. f77 nBos d 8 Casein Bos spp. f78 Bovine lactoferrin Bos spp. f334 f24 Shrimp rPen a 1 Tropomyosin Penaeus aztecus f351 f3: Fish (cod) rCyp c 1 Parvalbumin Cyprinus carpio f355 rGad c1 Parvalbumin Gadus morhua f426

Occupational allergens k82: Hevea brasiliensis rHev b 1 Hevea brasiliensis k215 rHev b 3 Hevea brasiliensis k217 rHev b 5 Hevea brasiliensis k218 rHev b 6.01 Hevea brasiliensis k219 rHev b 6.02 Hevea brasiliensis k220 rHev b 8 Profilin Hevea brasiliensis k221 rHev b 9 Enolase Hevea brasiliensis k222 rHev b 11 Hevea brasiliensis k224 Enzymes nAna c 2 Bromelin Ananas comosus k202 nApi m 1 Phospholipase A2 Apis mellifera k203 nCar p 1 Papain Carica papaya k201 nGal d 4 Lysozyme Gallus spp. k208 α-amylase From Aspergillus oryzane k87 Alkalase From Bacillus sp. k205 Maxatase From Bacillus licheniformis k204 Pepsin From Sus scrofa k213 Savinase From Bacillus sp. k206 Carbohydrate Determinants (CCD) Bromelin k202 CCD; MUXF3 (from bromelin) Ro214 r = Recombinant n = Natif

Patient: Nom : Mister Albert Gik Adresse: Birch Street 1… Birth date: 01/01/1950

Date de demande: 19/11/2009Signature:

Docteur: Nom: Cavalier Etienne Adresse : CHU of Liège

June 2009

Some other components less useful in Belgium could be available.Don’t hesitate to ask to your Lab in case of specific clinical history.

In theory, this is possible…

Practically, …

ImmunoCAP© ISAC

• VBC Genomics (expert in microarrays) and Phadia (expert in allergy in-vitro diagnostic tests) have combined “innovative biochip technology” with “cutting-edge research in molecular allergology” to develop ImmunoCAP®ISAC.

– Harwanegg C et al, Expert Rev Mol Diagn 2004

– Modern multiplex in vitro diagnostic tool for the allergy specialist, based exclusively on allergen components.

– Miniaturized immunoassay platform for measurement of sIgE antibodies to over 40 common allergen sources in a single step.

– 103 purified natural or recombinant allergen components immobilized on a solid support.

– Run CRD assays rapidly and cost-effectively.

Immuno Solid-phase AllergenChip (ISAC)

Allergen-triplet afterfluorescence-reaction

Allergen molecule

Fluorescence-labeledsecondary anti-human

IgE

IgE-antibody (in the patient’s serum)

4 reaction sites

ImmunoCAP® ISAC : Version 103.

Proteins, Cross-reactions :

– 11 PR-10 :

(Bet v 1-like proteins) : Well represented, interest ++

– 4 nsLTP : Some more ?

– Which ones?

– 5 Profilins : So many profilins exist… Which ones are most representative

in each botanic family ?

– 5 Storage proteins

– 2 Parvalbumins : Important ! Which ones are most

representative?

– 4 Tropomyosins : Important !

– 5 serum albumins : Which ones are most

representative?

– Honey bee and paper wasp venoms :

Lack…

Immuno Solid-phase Allergen Chip (ISAC)

Validation of the method

• Method– Selection of 19 sera of patients (6 males/13 females)

+ sIgE (>0.10 kUI/L) for recombinant allergens (ImmunoCAP ®250 Phadia).

positive and negative sIgE for different kind of allergenic sources (latex, peanut, birch, Timothy grass, hazelnut, peach, soybean, cat, dog…).

In all, we compared the results of 157 sIgE measured in CAP (recombinant allergens) with the ISAC®.

We tested 2 samples with a high TotIgE rate (> 10.000 kU/L) on ISAC ®.

(rHev b 1; n=3, rHev b 3; n=3, rHev b 8; n=5), cross-reactive carbohydrate determinants (nAna c 2; n=2), soja (rGly m 4; n=4), peanut (rAra h 1; n=7, rAra h 2; n=7, rAra h3; n=5, rAra h 8; n=12), hazelnut (rCor a 8; n=3), Brazil nut (rBer e 1; n=1), peach (rPru p 1; n=11, rPru p 3; n=3), birch (rBet v 1; n=17, rBet v 2; n=11, rBet v 4; n=8), timothy grass (rPhl p 1; n=12, rPhl p2; n=2, rPhl p4; n=5; rPhl p 5; n=7, rPhl p 6; n=3, rPhl p11; n=1, rPhl p 12; n=5), Aspergillus fumigatus (rAsp f 6; n=2), Alternaria (rAlt a 1; n=3), cat (rFel d 1; n=7) or dog (rCan f 1; n=4, rCan f 2; n=2, rCan f 3; n=2).


• Results:

– 17 results out of the 122 measurements of positive sIgE in CAP® were found negative with ISAC ® (concordance of 86%).

– 2 results out of the 35 measurements of negative sIgE in CAP® were found positive with ISAC ® (concordance of 94%).

– Until a rate of 150.000kU/L of TotIgE, we didn’t observe any unspecific binding.


• Discussion– Concordance of all positive results was 86%.

Can we explain the discordances?– Cut-off of the ISAC ® higher (>0,30 USI) than cut-off of CAP ® (>0,10 kUA/L)– Many measurements on ISAC ® were between the range of 0,10 and 0,30 USI.

– Concordance of all negative results was 94%. 2 discordances concerned sera for which ISAC® found a positive Ara h1

and Ara h 3 (2 major allergens of peanut).– Clinical importance of those 2 allergens !– Look back the clinical anamnesis :

» The first patient had positive sIgE in CAP for Ara h 1 and Ara h 2, his oral provocation test (TPO) to peanut was positive.

- Anamnesis well correlated with true positive Ara h 3 with ISAC ®

» The 2nd patient had undergone one TPO for peanut which was suspiscious, sIgE Ara h 8 ++, LTP ++, CCD +, polysensitized patient and large food eviction (hazelnut, peanut, sea food…)

- Anamnesis correlated well with a possible true positive Ara h 1 with ISAC ®

Pollens : Cyn d 1, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6 et Ole e 1

Bet v 1 homologues (PR-10):

Bet v 1, Aln g 1, Cor a 1, Mal d 1, Pru p 1, Gly m 4, Ara h 8, Api g 1

Profilins :

Bet v 2, Ole e 2, Hev b 8, Mer a 1, Phl p 12

Species markers :

Gal d 1, Bos d lactoferrin, Fel d 1, Fel d 4, Can f 1, Mus m 1, Alt a 1

Tropomyosin : Der p 10

Der p 1, Der f 1, Der f 2, Der p 2, Eur m 2

Albert Gik

Thanks to CRD, we can explain :

Many food allergies due to sensitization to profilins, PR-10 proteins (OAS), tropomyosin (sea food), cow milk…

Many weeds, mites, pets… which explain perannual rhinitis exacerbated during pollination.

Conclusion (I)

• The way for the discover of sensitization profiles is open…

– Allergen extracts are still useful : In first intention, Don’t permit to conclude every time (multiple positivity of the tests, lack of

precision), sIgE for extract not really significant.

– CRD is in our hands. Sensitizations can cause diagnosis errors (and therapeutic errors too). Protein families have been described, similarities of structure (homology of amino-

acids or structural homology) Allergy to one protein can cause cross-reactions to other proteins of the same family. Recombinant allergens permit to focus (one or more) allergen(s) of one source:

– Available in CAP (and/or in ISAC) or to be developed…

Conclusion (II)

• Microarrays can help us, in the difficult cases :– 103 recombinant or native purified allergens in one

determination… Analytical validation of the ImmunoCAP© ISAC performed:

– Concordance : 86% (negative results) and 94% (positive results)

The ImmunoCAP© ISAC has a role to play in the diagnosis and the care of patients with complex sensitization profiles.

As this technique provides potentially unexpected results, the microarray determination has to be used in conjunction with the clinical findings.

• Difficult interpretation of the results : – Should be limited to specialists !

Thank you for your attention !!

Documents

CRD and technical validation of ImmunoCAP ISAC ImmunoCAP ISAC Customer days Phn. Biol. E. Cavalier Phn. R. Gadisseur 19/11/2009